原代小鼠肝脏细胞的高效分离纯化与培养

目的：建立一种能稳定获得高活力和高纯度原代小鼠肝脏细胞的分离、纯化及培养方法。方法：应用改良的Seglen 二步法原位灌注和机械离心分离肝脏细胞,并用改良的高糖DMEM培养基进行培养。台盼蓝拒染法检测接种时肝脏细胞的存活率,倒置显微镜动态观察肝脏细胞形态变化,应用免疫荧光技术对肝脏细胞进行Albumin 染色。结果：每只小鼠可获取肝脏细胞的总产量平均为1.35× 10^6 / g体重,肝脏细胞存活率＞ 90%。倒置显微镜下观察贴壁前肝细胞直径为35.14 滋m± 4.35 滋m,肝脏细胞在接种后3 h基本完成贴壁;肝脏细胞接种后24h,所有肝脏细胞均强阳性表达成熟肝脏细胞标志物Albumin,肝细胞纯度＞ 95%。结论：改良的分离纯化及培养方法能稳定获得高产量、高活率及高纯度的小鼠肝脏细胞。     

原代小鼠肝脏细胞的高效分离纯化与培养*

摘要目的：建立一种能稳定获得高活力和高纯度原代小鼠肝脏细胞的分离、纯化及培养方法。方法：应用改良的Seglen 二步法原位灌注和机械离心分离肝脏细胞,并用改良的高糖DMEM培养基进行培养。台盼蓝拒染法检测接种时肝脏细胞的存活率,倒置显微镜动态观察肝脏细胞形态变化,应用免疫荧光技术对肝脏细胞进行Albumin 染色。结果：每只小鼠可获取肝脏细胞的总产量平均为1.35× 106 / g体重,肝脏细胞存活率＞ 90%。倒置显微镜下观察贴壁前肝细胞直径为35.14 滋m± 4.35 滋m,肝脏细胞在接种后3 h基本完成贴壁;肝脏细胞接种后24h,所有肝脏细胞均强阳性表达成熟肝脏细胞标志物Albumin,肝细胞纯度＞ 95%。结论：改良的分离纯化及培养方法能稳定获得高产量、高活率及高纯度的小鼠肝脏细胞。     

周细胞的原代培养

目的:探讨Wistar大鼠视网膜毛细血管周细胞(pericyte,PC)的原代培养和鉴定方法。方法:结合视网膜微血管的消化分离,采用含10%胎牛血清的DMEM培养基选择性培养PC,通过活细胞观察原代PC的形态、生长特性以及与血管碎片之间的关系,同时应用免疫细胞化学染色来鉴定PC。结果:选择性培养获得的PC的纯度达到95%以上,并能连续传代。该细胞呈长梭形或星芒状,漩涡或栅栏状生长,无接触性抑制,单核,偶见双核,核卵圆形,细胞浆丰富,α-SMA、PDGFR-β染色阳性。结论:通过对视网膜微血管的消化分离能够获得较为纯净的PC。     

人表皮细胞分离培养的研究

目的探索来源于人的表皮细胞的分离培养方法,为其进一步作为皮肤组织工程中的种子细胞或临床应用奠定前期研究基础。方法取3~9岁健康儿童包皮环切术后包皮,经分离酶处理分离真表皮,再将表皮以胰蛋白酶消化为细胞悬液,分别接种于有血清培养基DMEM和无血清培养基K-SFM中进行细胞培养,观察表皮细胞生长融合情况及克隆形成率。结果表皮细胞在DMEM和K-SFM培养液中均能融合成片,但在K-SFM中的融合成片时间明显短于在DMEM中所需时间;在K-SFM中2周时克隆形成率显著高于在DMEM中的克隆形成率。结论两步酶法分离表皮细胞接种于K-SFM中培养,是一种简便有效的表皮细胞分离培养方法。     

改良胶原酶消化法分离培养人原代髓核细胞

目的:优化人原代髓核细胞的体外分离培养方法,为椎间盘退变的防治研究提供种子细胞。方法:无菌环境中摘取人椎间盘髓核组织,采用多次胶原酶消化法分离提取原代人髓核细胞,置于5%CO2培养箱中37℃恒温培养,倒置相差显微镜中观察细胞形态,采用MTT法绘制细胞生长曲线,甲苯胺蓝染色法检测髓核细胞内蛋白多糖的表达情况,细胞免疫荧光染色法检测Ⅱ型胶原蛋白表达情况。结果:本研究中获得的细胞形态不规则,呈梭形或多角形,原代细胞48 h内贴壁,培养第8天左右细胞融合度可达90%,第三代细胞12 h内即可贴壁,生长至融合90%约需5d。甲苯胺蓝染色及细胞免疫荧光染色均阳性,提示所得细胞具有分泌蛋白多糖及Ⅱ型胶原蛋白的功能。结论:改良胶原酶消化法可获得大量纯净的人髓核细胞,提高培养效率,原代及传代细胞具备类软骨细胞表型,且活性及功能均较为稳定,可作为椎间盘组织工程研究的种子细胞。     

氯通道在肾脏的表达及其功能

随着膜片钳和分子克隆技术的应用,人们在肾脏发现了多种氯通道。这些通道参与许多生理过程,包括膜电位形成、细胞容积调节以及盐的吸收和排泄等。确定氯通道在肾小管和集合管上皮细胞的基侧膜和管腔膜的分布情况以及这些通道的生理功能是近年来的研究热点。最近,由于特异性抗体的应用和遗传学研究方法的建立,使这方面的研究取得了很大进展。本文就这方面的研究进展作一综述。     

大鼠髓核细胞原代培养及表型鉴定

目的:探讨Ⅱ型胶原酶联合透明质酸酶消化分离培养髓核细胞及免疫细胞化学表型鉴定的可行性。方法:无菌条件下分离SD大鼠凝胶状髓核,采用Ⅱ型胶原酶联合透明质酸酶消化分离髓核细胞并连续培养,倒置相差显微镜下观察,随后进行免疫细胞化学染色检测不同代次髓核细胞HIF-1、Ⅰ、Ⅱ型胶原、MMP2及蛋白聚糖的表达情况,并给予MTT法测定髓核细胞生长曲线。结果:Ⅱ型胶原酶联合透明质酸酶分离培养原代髓核细胞需要12 d左右贴壁,达95%融合需要34 d,而传代髓核细胞贴壁速率明显增快至10 h,且其倍增时间约为2.5 d;免疫细胞化学显示髓核细胞均表达HIF-1、Ⅰ、Ⅱ型胶原、MMP2和蛋白聚糖,且随着髓核细胞传代其HIF-1α、HIF-1β、Ⅰ型胶原及MMP2表达均增加,但Ⅱ型胶原表达降低,而蛋白聚糖表达无明显差异;MTT法显示随着髓核细胞传代其增殖有所减缓。结论:Ⅱ型胶原酶联合透明质酸酶可成功分离髓核细胞,提高培养效率,且HIF-1α、HIF-1β、Ⅰ、Ⅱ型胶原及MMP2可作为髓核细胞表型分子用于髓核细胞的鉴定。     

正常人肝脏星形细胞的分离,培养及鉴定

参考Friedman等分离人肝脏星形细胞(HSC)的方法,采用校正密度的淋巴细胞分离液进行一步梯度离心,成功地分离得到了正常人HSC。HSC的收获量约为1×10~7个/10克肝脏组织,存活率在95％以上,纯度达85％以上。传代后纯度接近100％。对人HSC的标志物进行检测发现,结蛋白不宜作为鉴定初分离的及原代培养初期人HSC纯度的指标,而α-平滑肌肌动蛋白可作为鉴定激活的人HSC的可靠指标。     

小胶质细胞纯化分离培养方法的改良

目的改良现有的小胶质细胞纯化分离培养方法,建立稳定简便的培养模型。方法利用盐酸利多卡因注射液代替机械振摇纯化分离小胶质细胞;利用CD11b/c(OX42)免疫细胞化学的方法对分离的小胶质细胞纯度进行鉴定,同时观察小胶质细胞形态及活化指标NFкBp65的表达情况;利用流式细胞仪,结合细胞计数及MTT细胞活力测定检测纯化分离后小胶质的增殖情况。结果改良的方法可稳定的获得1.2×106个/培养瓶(75cm2,250ml)的小胶质细胞,纯度达到98%,存活率≥95%,形态上以阿米巴样为主,继续培养3-5d,约半数细胞可转变为静止状态。NFкBp65免疫细胞化学染色为胞浆表达。流式细胞仪检测结合细胞计数及MTT细胞活力检测结果显示,体外纯化培养的小胶质细胞多位于G0/G1期,培养过程中未出现明显的增殖。结论改良的方法易于操作,产量多,纯度高。为体外小胶质细胞进一步研究提供了基础。     

葡萄糖浓度波动对原代培养的大鼠血管细胞和肾细胞的影响

探讨葡萄糖浓度波动对体外培养的原代大鼠血管细胞和肾细胞的影响。取SD大鼠的主动脉和肾脏进行血管细胞和肾细胞的体外原代培养,每种细胞均分为6组:正常对照组、持续高糖组、持续低糖组、波动组I、波动组II、波动组III。实验24h后,测定细胞乳酸脱氢酶(LDH)的泄漏率,细胞液中的β-N-乙酰氨基葡萄糖苷酶(NAG)的活力,细胞内还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的活力。结果表明葡萄糖浓度波动各组均能对大鼠血管细胞和肾细胞造成损伤,使细胞外液LDH、NAG的泄漏量明显增加,细胞内GSH、SOD活力明显减少,与持续高糖组和持续低糖组比较差异显著(P<0.001)。且葡萄糖浓度波动对肾细胞的损伤比血管细胞更为明显。说明葡萄糖浓度波动能够导致大鼠血管细胞和肾细胞的损伤,并且其损伤远远大于持续高糖或持续低糖的单独作用效果,损伤的结果与低糖作用细胞的时间呈正相关,在相同的损伤条件下肾细胞比血管细胞对葡萄糖浓度波动更为敏感,损伤更为严重。     

Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Immortalized renal proximal and collecting duct cell lines derived from transgenic mice harboring L-type pyruvate kinase promoters as tools for pharmacological and toxicological studies

Targeted oncogenesis in transgenic mice, where an oncogene is placed under the control of the regulatory sequences of a cell-specific gene, has been used to derive lines of differentiated kidney epithelial cells derived from proximal or distal tubules or from the collecting duct. These renal cell lines were obtained from kidneys of transgenic mice harboring the large-T and little-t antigens placed under the control of regulatory sequences of the L-type pyruvate kinase gene. This review summarizes the main properties of these differentiated cell lines, which are usefulex vivo cell systems for pharmacological and toxicological studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Urea transport in freshly isolated and cultured cells from rat inner medullary collecting duct

Summary Regulation of urea transport by vasopressin in inner medullary collecting duct (IMCD) cells is thought to be important for the urinary concentrating mechanism. Isolated tubule perfusion studies suggest the existence of a saturable urea carrier. We have measured¹⁴C-urea efflux in IMCD cells which were freshly isolated and grown in primary culture. Cells were isolated from rat papilla by collagenase digestion and hypotonic shock. In suspended cells,¹⁴C-urea efflux (J _urea from loaded cells was exponential with time constant 59±3 sec (sem,n=6, 23°C).J _urea had an activation energy of 4.1 kcal/mole and was inhibited 42±7% by 0.25mm phloretin and 30–40% by the high affinity urea analogues dimethylurea and phenylurea.J _urea was increased 40–60% by addition of vasopressin (10^–8 m) or 8-bromo-cAMP (1mm); stimulatedJ _urea was inhibited 55±8% by the kinase A inhibitor H-8. Phorbol esters and epidermal growth factor did not alterJ _urea. IMCD cells grown in primary culture were homogeneous in appearance with>fivefold stimulation of cAMP by vasopressin. The exponential time constant for urea efflux was 610±20 sec (n=3).J _urea was not altered by vasopressin, cAMP or phloretin. Another function of in vivo IMCD cells, vasopressin-dependent formation of endosomes containing water channels, was absent in the cultured cells. These results demonstrate presence of a urea transporter on suspended IMCD cells which is activated by cAMP and inhibited by phloretin and urea analogues. The urea transporter and its regulation by cAMP, and cAMP-dependent apical membrane endocytosis, are lost after growth in primary culture.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Effect of dDAVP on basolateral cell surface water permeability in the outer medullary collecting duct

We report a novel approach for assessing the volume of living cells which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The aim of this study was to evaluate the short-term effect of vasopressin on basolateral cell surface water permeability in the outer medullary collecting duct (OMCD). The permeability of the basolateral cell membrane was determined in the tubules where the apical membrane was blocked with oil injected into the lumen. The apparent coefficient of water permeability (P _f) was evaluated by measuring the cell swelling after the step from hypertonic to isotonic medium (600 mosm to 300 mosm). Desmopressin (dDAVP) induced an increase of the basolateral P _f from 113.7±8.5 μm/s in control cells to 186.6±11.4 μm/s in micro-dissected fragments of the OMCD incubated in vitro (10⁻⁷ M dDAVP, 30 min at 37 °C) (P<0.05). Mercury caused pronounced inhibition of basolateral water permeability (26.0±6.9 μm/s; P<0.05). The effect of mercury (1.0 mM HgCl₂) was reversible: after washing the fragments with PBS for 20 min, P _f values were restored to the control levels (125.0±9.5 μm/s). The results of the study indicate the existence of a mechanism controlling the osmotic water permeability of the basolateral cell membrane in the OMCD epithelium.     

The Cell Adhesion Molecule L1 Is Developmentally Regulated in the Renal Epithelium and Is Involved in Kidney Branching Morphogenesis

We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequencing of three polypeptides identified the antigen as the neuronal cell adhesion molecule L1, a member of the immunoglobulin superfamily. The kidney isoform showed a deletion of exon 3. L1 was expressed in the mesonephric duct and the metanephros throughout CD development. In the adult CD examined by electron microscopy, L1 was not expressed on intercalated cells but was restricted to CD principal cells and to the papilla tall cells. By contrast, L1 appeared late in the distal portion of the elongating nephron in the mesenchymally derived epithelium and decreased during postnatal development. Immunoblot analysis showed that expression, proteolytic cleavage, and the glycosylation pattern of L1 protein were regulated during renal development. L1 was not detected in epithelia of other organs developing by branching morphogenesis. Addition of anti-L1 antibody to kidney or lung organotypic cultures induced dysmorphogenesis of the ureteric bud epithelium but not of the lung. These results suggest a functional role for L1 in CD development in vitro. We further postulate that L1 may be involved in the guidance of developing distal tubule and in generation and maintenance of specialized cell phenotypes in CD.     

Apico-basal osmotic gradient induces transcytosis in cultured renal collecting duct epithelium

Summary The present experiments report the existence of an apico-basal plasma membrane shuttle in cultured renal collecting duct principal cell epithelium. Apical and basal perfusion under isotonic conditions, 290 mosm phosphate-buffered saline (PBS), has no effect on the shape of the epithelium. In contrast, gradient perfusion bf the epithelium with 75 mosm PBS on the apical side and 290 mosm PBS on the basal side for 10 min alters the morphology of the epithelium by causing the originally columnar epithelial cells to become lower, the intercellular spaces to dilate, and the intracellular vesicles to enlarge. Perfusion of the epithelium with isotonic PBS in the presence of electron-dense cellular markers such as gold-coupled GP_CDI antibody, recognizing a glycoprotein in the plasma membrane of collecting duct cells (W.W. Minuth, G. Lauer, S. Bachman and W. Kriz,Histochemistry 80:171–182, 1984), cationized ferritin (CF), horseradish peroxidase (HRP) and native ferritin (NF) for 10 min reveals their binding at the apical plasma membrane. Little endocytosis is observable. However, after labeling the luminal side by the cellular markers and following exposure to apical hypotonicity, 75 mosm PBS for 10 min, endocytosis of all markers is enhanced to a high degree. Furthermore, the gold-coupled GP_CDI antibody and cationized ferritin are transported within vesicles unidirectionally through the epithelium and are exocytosed at the basolateral aspect, indicating the retrieval and possible translocation of apical plasma membrane. In contrast, volume markers such as NF and HRP are also endocytosed under osmotic gradient exposure, but are not seen to be transcytosed. Therefore, the function of this membrane pathway seems not to be related to water reabsorption, but may be part of a cellular response as protection against the osmotic gradient.